The present invention relates to expression systems and methods for detecting protein—protein interactions in cells by expressing in the cells small peptide molecules and monitoring their affect on cellular signal transduction pathways. As such, the present invention enables the identification and selection of small molecules which can be used for the production of new pharmaceutical lead compounds, which can, in turn, be used for drug development and for diagnostics and research studies. The expression systems and methods of the present invention can also be used to identify novel constituents of intra cellular transduction pathways which can be the subject of further research and study.
Control of a variety of cellular processes, including, but not limited to, growth, differentiation and function, rely upon protein—protein interactions. Protein-protein interactions are intrinsic to virtually every cellular process, such interactions control, for example, cell division processes, protein expression in cells, etc.
A signal transduction cascade is a process involving the conversion of a cell's interaction with an external regulators, such as hormones, cytokines and the like, to a specific internal response, such as upregulation or down regulation of the expression of a specific gene or genes.
A signal transduction cascade initiates at the outer surface of the cell membrane, there, an external stimulus which can be molecular (e.g., a polypeptide), or physical (e.g., light, shear stress, etc.), initiates an intra-cellular cascade of protein—protein interaction(s) and/or enzymatic reactions. This cascade ultimately leads to the upregulation or down regulation of the expression of specific gene or genes within the cell, which expression characterizes the cellular response to the external stimulus.
Signal transduction pathways are effected by protein complexes which are formed in different compartments of the cell according to the targeted signals of the proteins (Pawson T. and Scott J D., Science 1999, 278:2075–2079). Signal transduction pathways are tightly regulated processes, and many disorders and diseases are characterized in dysfunctional cellular transduction pathways.
For example, many cancer cells are characterized by dysfunctional signal transduction pathway components, which pathways regulate for example, cell proliferation.
Thus, it is of great interest to develop methods which can be used to identify polypeptides which interfere with cellular process, and as such, to control, alleviate or treat such disorders and diseases. Such polypeptides can then be used to develop novel drugs or to be used as drug targets or to identify new genes.
Currently, studies are being conducted in an effort to isolate inhibitors to cellular processes based upon either deciphering protein structure and function or studying the components involved in protein—protein interactions. Several methods with which protein—protein interactions can be studied are known in the art. The most common biochemical methods employed include, protein affinity chromatography, affinity blotting, co-immune-precipitation and protein cross linking. Molecular biology methods have been developed and include epitope tagging, two hybrid systems, three hybrid systems and phage display libraries. In addition, genetic methods which include the uncovering of genetic mutants have also been employed. For review see Phizicky E M and Fields S. Microbiol. Rev. 1995, 59:94–123.
Following, is a brief description of each of the various methods for studying protein—protein interactions commonly employed in the art.
Protein Tag:
The protein tag method involves the generation of a fusion (translational fusion of DNA sequences) between a peptide tag sequence and a defined protein sequence, so as to form a chimera protein. This chimera can serve as a tool to isolate proteins which interact with the defined protein, by using the tag sequence as an indicator for the presence of interaction. For example, antibodies generated against the tag peptide enable the visualization of the protein or protein complex on a blot or to affinity purify specific protein complexes via affinity columns or immuno-precipitation (Skolnik et al. Cell, 1991, 65:83–90).
Phage Display Library:
Phage particles consist of a nucleic acid molecule surrounded by a proteinaceous coat which enables the phage to interact with, and infect, host bacteria. Filamentous phages, such as M13, can express a fusion protein bearing a foreign peptide on the coat surface by infecting a bacterial host such as E. Coli (Smith G P, Science 1985, 228:1315–1317). DNA sequences coding for protein or peptide of interest are translationally fused to the N terminal of the gene encoding one of the phage coat proteins (e.g., V3 or V8 in M13). If the translational fusion does not interfere with the life cycle of the bacteriophage, the modified phage particle will express a chimeric coat protein which displays the foreign peptide or protein of interest. Phage particles “displaying” the foreign peptide or protein on their surface can be selected for by immune-affinity purification. Phage display libraries can be prepared by constructing a collection of phage particles each capable of displaying a different foreign peptide.
Random peptide phage display libraries have proved to be a useful tool for identifying the protein constituents of various protein—protein interaction reactions (Parmley, S. F. And G. P. Smith, 1989, Adv Exp Med Biol, 251:215–218; Scott, J. K. and G. P. Smith 1990, Science 249:386–390; Winter, J. 1994, Drug and Dev Res 33:71–89). Such libraries have also been used to define epitopes of monoclonal and polyclonal antibodies and to define the specificity of extracellular and cytosolic receptors (Devlin et al., 1990, Science 249:404–406; Doorbar J. and G Winter, 1994 J Mol Biol 244:361–369; Kay, B. K. 1995, Perp Drug disc 2:251–268).
Two-Hybrid System:
The two-hybrid system uses transcriptional activity as a measure of protein—protein interactions. This system takes advantage of the modular nature of many site specific transcriptional activators. Many transcriptional activators consist of a DNA binding domain and a transcriptional activation domain (Chein, C T. et al. Proc. Natl. Acad. Sci. 1991, 88:9578–9582, Fields S. and Song O K. 1989, Nature, 1989, 340:245–246, Fields S. and Sternglanz R. Trends. Genet. 10:286–292). The DNA binding domain serves to target the activator to a specific gene to be expressed, while the activation domain binds other proteins of the transcriptional machinery to thereby initiate transcription. The two domains of the transcriptional activator need not be covalently linked but simply brought into proximity to initiate transcription. The two domains of the transcriptional activator can be brought into proximity by a pair of interacting proteins. This is achieved by constructing two hybrids, a first hybrid in which the DNA binding domain of the transcriptional activator fused to a first protein, and a second hybrid in which the transcription activation domain of the transcriptional activator is fused to a second protein. These two hybrids are overexpressed in a cell containing one or more reporter genes under the control of a cis acting element that is known to bind the DNA binding domain. If the first and second proteins interact, the domains of the activator are brought into proximity and the reporter gene is activated. Since the two hybrid system involves the utilization of nucleus functioning transcriptional activator this system is limited to interactions which can occur in the nucleus, thus preventing its use with certain extracellular proteins. Numerous variations on the two hybrid method have been proposed, see for example the reverse two hybrid described in U.S. Pat. No. 5,965,368 to Vidal et al.
Three Hybrid System:
The three hybrid system can be used to analyze interactions between three distinct components. This system is typically used to detect and analyze RNA protein interactions in which the binding of bifunctional RNA to each of two hybrid proteins activates transcription of a reporter gene in-vivo. This binding relies on the physical properties of the RNA and protein and not on their natural biological activities (SenGupta D J. et al., 1996, Proc. Natl. Acad. Sci. 93:8496–8501).
Although the above described methods enable the detection and subsequent isolation of interacting proteins, these methods are generally used to detect interactions in which one component is a known component, and as such in cases where a protein which interacts with transduction pathway proteins is sought, hybrids must be constructed for all of the proteins involved in the pathway.
There is thus a widely recognized need for, and it would be highly advantageous to have, an in-situ method for detecting and isolating polypeptides which regulate transduction pathways devoid of the above limitations.